Method for improving quality of whole-plant corn silage feed by using litsea cubeba residue

Abstract

The application provides a method for improving the quality of whole corn silage feed by using Litsea cubeba residues, and belongs to the technical field of feed processing. The whole corn is crushed, and Litsea cubeba residues are added to obtain mixed raw materials, the addition amount of the Litsea cubeba residues is 1% to 4% of the fresh weight of the crushed whole corn; the mixed raw materials are loaded into a silage container, compacted, sealed, and subjected to silage fermentation to obtain the whole corn silage feed. The Litsea cubeba residues are the core residues remaining after the extraction of essential oil from Litsea cubeba fruits. In the application, the Litsea cubeba residues are used as a feed additive, and the effects of adding different proportions of the Litsea cubeba residues on the nutritional components of the whole corn silage feed, the silage fermentation quality, the in vitro rumen fermentation characteristics, the microbial community structure, and the dynamic changes of material metabolism and microorganisms during aerobic exposure are systematically studied, so as to provide data support for the reasonable use of the Litsea cubeba residues and theoretical basis for analyzing the related mechanism of the aerobic stability of the whole corn silage.

Description

Technical Field

[0001] This invention belongs to the field of feed processing technology, and in particular relates to a method for improving the quality of whole-plant corn silage using Litsea cubeba residue. Background Technology

[0002] Litsea cubeba ( Litsea cubeba Litsea cubeba, also known as mountain pepper, is a deciduous small tree or shrub belonging to the genus Litsea in the Lauraceae family. It grows to a height of 8-10 meters and is mainly distributed in provinces south of the Yangtze River in China, extending to Tibet. It is a unique aromatic economic tree species in my country. The fruit of Litsea cubeba is rich in essential oils, with citral content as high as 60%-90%, far exceeding that of other foreign varieties. It is an important natural fragrance and pharmaceutical chemical raw material. my country has a vast territory and excellent natural environmental conditions, resulting in extremely rich woody oil resources. According to statistics, the planting area of ​​Litsea cubeba in 2018 was 14,400 hectares. 2 Annual production of essential oils is 140,100 tons.

[0003] After extracting essential oils from Litsea cubeba fruit using methods such as steam distillation, a large amount of solid residue (hereinafter referred to as "Litsea cubeba residue") is produced. Currently, this residue is usually discarded or incinerated, causing not only environmental pollution but also a significant waste of biomass resources. Studies have shown that Litsea cubeba residue not only retains some plant essential oil components but also contains abundant crude protein, crude fiber, minerals, and other nutrients, possessing high potential for feed development. However, current research reports on the resource utilization of Litsea cubeba residue in the feed field are still very limited.

[0004] Whole-plant corn silage is a widely used roughage processing method in animal husbandry, offering advantages such as high nutrient retention, good palatability, and long-term storage. However, once opened from the silo and exposed to air, whole-plant corn silage is highly susceptible to secondary fermentation due to the proliferation of aerobic microorganisms (such as yeast and mold). This results in increased feed temperature, elevated pH, and significant nutrient loss; in severe cases, it can even mold and spoil, rendering the silage unusable. This phenomenon, known as "poor aerobic stability," is one of the key bottlenecks restricting the widespread application of whole-plant corn silage.

[0005] Existing research has reported that adding Litsea cubeba essential oil can inhibit mold and slow down the loss of crude protein and amino acids in silage corn (Zhang Fujin et al., 2022). However, Litsea cubeba essential oil is relatively expensive, making its large-scale application in silage production economically unfeasible. More importantly, there is currently a lack of systematic research and publicly available technical solutions regarding whether Litsea cubeba residue, an inexpensive byproduct of essential oil extraction, can replace expensive essential oil in improving the quality of whole-plant corn silage, especially its aerobic stability, and what its mechanism of action is.

[0006] Therefore, developing a method to improve the aerobic stability of whole-plant maize silage by utilizing the waste resource of Litsea cubeba residue can not only realize the resource utilization of agricultural and forestry by-products and reduce environmental pollution, but also effectively improve the quality of silage and extend its shelf life after aerobic exposure, which is of great significance for promoting the green and sustainable development of animal husbandry. Summary of the Invention

[0007] To address the aforementioned problems in the prior art, this invention provides a method for improving the quality of whole-plant maize silage using Litsea cubeba residue. This invention uses Litsea cubeba residue as a feed additive and systematically studies the effects of adding different proportions of Litsea cubeba residue to whole-plant maize silage on its nutritional components, silage fermentation quality, rumen in vitro fermentation characteristics, microbial community structure, and dynamic changes in metabolism and microorganisms during aerobic exposure. This provides data support for the rational utilization of Litsea cubeba residue and a theoretical basis for elucidating the mechanisms related to the aerobic stability of whole-plant maize silage.

[0008] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of the present invention: This invention provides a method for improving the quality of whole-plant corn silage using Litsea cubeba residue, comprising the following steps: (1) After crushing the whole corn plant, add the Litsea cubeba residue to obtain a mixed raw material, wherein the amount of Litsea cubeba residue added is 1%-4% of the fresh weight of the crushed whole corn plant; (2) The mixed raw materials are loaded into a silage container, compacted and sealed, and fermented to obtain the whole-plant corn silage.

[0009] Furthermore, the Litsea cubeba residue mentioned in step (1) is the residue remaining after extracting essential oil from Litsea cubeba fruit. The Litsea cubeba residue contains 95.18% dry matter (DM), 13.01% crude protein (CP), 34.74% crude fat (EE), 44.06% neutral detergent fiber (NDF), and 11.08% acid detergent fiber (ADF).

[0010] Furthermore, the amount of Litsea cubeba residue added in step (1) is 2% of the fresh weight of the whole corn plant after crushing.

[0011] Furthermore, the silage fermentation described in step (2) is carried out for 60 days.

[0012] Furthermore, after the silage fermentation in step (2) is completed, it is subjected to aerobic exposure for 0-5 days, wherein the aerobic exposure for 0 days is when the silage is opened.

[0013] Furthermore, the quality of the whole-plant corn silage includes aerobic stability, nutritional components, fermentation quality, and rumen in vitro fermentation characteristics.

[0014] Furthermore, the method for determining the nutritional components and rumen in vitro fermentation characteristics is as follows: the whole-plant corn silage is blanched and then dried, and then the nutritional components and rumen in vitro fermentation characteristics are determined. The blanching temperature was 105℃, and the time was 20 min; The drying temperature is 65°C and the time is 48 hours.

[0015] Furthermore, the method for determining the fermentation quality is as follows: the silage exposed to aerobic conditions is prepared into an extract, and the pH and fermentation quality are measured.

[0016] The beneficial effects of this invention compared to the prior art are as follows: This invention, by adding an appropriate amount of Litsea cubeba residue to whole-plant corn silage, effectively inhibits the growth and reproduction of harmful microorganisms such as yeast and mold during aerobic exposure, delays the secondary fermentation process of the silage, and thus significantly improves its aerobic stability and extends its shelf life after opening the silo. Experimental results show that the optimal aerobic stability is achieved when the amount of Litsea cubeba residue added is 2% of the fresh weight of the whole-plant corn.

[0017] The addition of Litsea cubeba residue promotes the formation of acetic acid during silage fermentation. Acetic acid is a key indicator of aerobic stability in silage and can effectively inhibit the growth and reproduction of yeast and mold. When the addition amount of Litsea cubeba residue is 2% of the fresh weight of the whole corn plant, the acetic acid content is significantly higher than other addition ratios, which is the direct reason for its optimal aerobic stability. Moreover, the appropriate addition of Litsea cubeba residue also promotes the full progress of silage fermentation, allowing water-soluble carbohydrates (WSCs) to be fully utilized and significantly reducing the residual amount, thereby reducing the substrate source available for aerobic putrefaction and fundamentally reducing the risk of secondary fermentation. Experimental results show that when the addition amount exceeds 2%, aerobic stability actually decreases. Excessive addition of Litsea cubeba residue inhibits the activity of heterotrophic fermentation bacteria that produce acetic acid, leading to a significant decrease in acetic acid content; at the same time, excessive addition inhibits the overall fermentation efficiency, resulting in insufficient utilization of WSCs and a large amount of residual WSCs, providing substrate for aerobic putrefaction. This invention accurately determines the optimal addition ratio, avoids the negative effects of excessive addition, and achieves efficient utilization of resources.

[0018] In this invention, the amount of Litsea cubeba residue added is strictly controlled to maintain the lactic acid content at a moderate level. Lactic acid helps with rapid acidification in the early stages of silage, but excessive lactic acid content can inhibit the activity of heterotrophic fermentation bacteria that produce acetic acid. This invention achieves optimized regulation of lactic acid content by controlling the addition ratio of Litsea cubeba residue, maintaining a good fermentation microecological balance and creating favorable conditions for the efficient production of acetic acid.

[0019] The *Litsea cubeba* residue used in this invention is the solid residue remaining after extracting essential oil from *Litsea cubeba* fruit, belonging to the category of agricultural product processing byproducts. *Litsea cubeba* residue not only retains some plant essential oil components but also possesses high nutritional value, making it suitable for use as a feed resource. This invention transforms this waste into a valuable resource, developing it into a highly efficient and inexpensive silage additive. This significantly reduces the production cost of silage (compared to directly adding *Litsea cubeba* essential oil) and opens up new avenues for the comprehensive utilization of byproducts from the *Litsea cubeba* processing industry, yielding excellent economic, social, and ecological benefits. Furthermore, the method of this invention is simple, requiring only the addition of *Litsea cubeba* residue to whole-plant corn silage, without the need for additional equipment investment or complex processes, making it easy to promote and apply on a large scale in feed production and livestock enterprises. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a flowchart of the sequencing experiment for the dynamic changes in silage metabolism and microorganisms during aerobic exposure, as described in this invention. Figure 2 This is a diagram showing the functional prediction and analysis of the microbial community during the aerobic exposure process of silage, reflecting the dynamic changes in silage metabolism and microorganisms. Figure 3 This is a LC-MS non-targeted metabolic grouping percentage stacked bar chart of corn silage from Litsea cubeba residue during the aerobic exposure process of the present invention to illustrate the dynamic changes in silage metabolism and microorganisms. Figure 4 The importance of LC-MS non-targeted metabolic random forest features of corn silage made from Litsea cubeba residue during aerobic exposure is shown in the dynamic changes of silage metabolism and microorganisms in the present invention. Figure 5 A bubble diagram illustrating the enrichment analysis of the KEGG metabolic pathway in the dynamic changes of silage metabolism and microorganisms during aerobic exposure, as presented in this invention. Figure 6 This is a bar chart showing the relative distribution of phylum-level microbial communities in the dynamic changes of silage metabolism and microorganisms during aerobic exposure, as described in this invention. Figure 7 This is a bar chart showing the relative distribution of bacterial communities at the genus level in terms of the dynamic changes of silage metabolism and microorganisms during aerobic exposure in accordance with the present invention. Figure 8This is a bar chart showing the relative distribution of fungal communities at the phylum level in terms of the dynamic changes of silage metabolism and microorganisms during aerobic exposure in this invention. Figure 9 This is a bar chart showing the relative distribution of fungal communities at the genus level in terms of the dynamic changes of silage metabolism and microorganisms during aerobic exposure, as presented in this invention. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example A method for improving the quality of whole-plant maize silage using Litsea cubeba residue includes the following steps: (1) The harvested whole corn plant was crushed using a crusher and shredder, mixed evenly and set aside. A single-factor experiment was conducted, and 0% (control group, group A), 1% (group B), 2% (group C), 3% (group D) and 4% (group E) of Litsea cubeba residue were added to the crushed whole corn plant and mixed evenly to obtain 5 groups of mixed raw materials with different amounts of Litsea cubeba residue. The amount of Litsea cubeba residue added was based on the fresh weight of the whole corn plant.

[0028] (2) Take 710 g of the above 5 groups of mixed raw materials with different amounts of Litsea cubeba residue and put them into 1 L silage buckets, compact and seal them. Set up 4 replicate experiments for each group. After 60 days of silage fermentation, aerobic exposure was carried out for 5 days to obtain 5 groups of whole plant corn silage with different amounts of Litsea cubeba residue.

[0029] Measurement results 1. Nutritional components 200 g of the above whole-plant corn silage was blanched in a 105℃ forced-air drying oven for 20 min, and then dried in a 65℃ oven for 48 h. The dried sample was taken out, pulverized by a laboratory grinder, and passed through a 40-mesh sieve. The dry matter (DM), crude protein (CP), crude fat (EE), and crude ash (Ash) of the pulverized silage were determined according to the corresponding official methods in AOAC (2005) "Official Methods of Analysis" (18th ed.). Among them, DM: loss on drying method, Ash: 550℃ muffle furnace ashing method, CP: Kjeldahl nitrogen determination method, N×6.25, and EE: Soxhlet extraction method. The acid detergent fiber (ADF) and neutral detergent fiber (NDF) content of crushed silage were determined according to the Van Soest washing fiber method and using an ANKOM fiber analyzer (ANKOMA200i Fiber Analyzer, Ankom Technology, Macedon, NY) and ANKOM F57 filter bags. The water-soluble carbohydrate content was determined using the anthrone-sulfuric acid colorimetric method. Results are expressed on a dry basis (%DM) and are shown in Table 1. In Table 1, SEM represents the error between the sample mean and the population true mean, and P-values ​​represent probability values.

[0030] Table 1

[0031] Note: Different lowercase letters in the upper right corner of the data indicate significant differences between groups (P<0.05), while the same lowercase letter indicates no significant differences between groups (P>0.05).

[0032] Table 1 shows that the DM content in group D was significantly higher than that in groups A, B, and C (P<0.05), and the DM content in group E was also significantly higher than that in groups A, B, and C (P<0.05). The DM content in control group A was significantly lower than that in other treatment groups (P<0.05). The CP content in control group A was significantly lower than that in other treatment groups (P<0.05). The EE content in control group A was significantly lower than that in other treatment groups (P<0.05). For acid detergent fiber, the content in control group A was significantly lower than that in other treatment groups (P<0.05). The WSC content in groups D and E was significantly higher than that in group C (P<0.05). Excessive addition of Litsea cubeba residue may have inhibited overall fermentation efficiency, resulting in underutilized sugars and higher residue levels, providing substrate for aerobic putrefaction. Furthermore, the WSC content in group C was significantly lower than that in other groups (P<0.05), reducing the substrate source for aerobic putrefaction.

[0033] 2. Fermentation quality Take 20 g of whole-plant corn silage from each of the five groups with different amounts of Litsea cubeba residue, add 180 mL of distilled water, stir thoroughly, and crush using a juicer for 1 min. Filter the mixture through four layers of gauze and qualitative filter paper, collect the leachate after filtration through the qualitative filter paper, and dilute to 200 mL with distilled water. Measure the pH value using a pH meter (Mettler-Toledo DELTA320 pH meter). Then filter the diluted leachate through a 0.45 μm filter membrane and determine the volatile fatty acid (VFA) and lactic acid content of the filtrate using a gas chromatograph (Agilent 8860). The volatile fatty acids included acetic acid, propionic acid, butyric acid, and valeric acid (not detected). The analytical conditions were: wavelength 214 nm, C18 column, column temperature 30℃, mobile phase 0.02 mol / L KH2PO4 and H3PO4, pH 2.37, flow rate 1 mL / min, and injection volume 10 mL / min. μL; ammonia nitrogen was determined using the phenol-sodium hypochlorite colorimetric method. The results are shown in Table 2.

[0034] Table 2

[0035] Note: Different lowercase letters in the upper right corner of the data indicate significant differences between groups (P<0.05), while the same lowercase letter indicates no significant differences between groups (P>0.05).

[0036] Table 2 shows that the pH value of group B was significantly higher than that of other treatment groups (P<0.05). There was no significant difference in acetic acid content among the treatment groups (P>0.05), but group C had a higher acetic acid content than the other groups. Acetic acid is a key indicator of aerobic stability in silage and effectively inhibits the growth of yeast and mold, which is the direct reason why group C had the best aerobic stability. Group C had a moderate lactic acid content, significantly higher than groups A, B, and E (P<0.05). Lactic acid helps with rapid acidification, but excessive amounts can inhibit heterofermentative bacteria that produce acetic acid. Groups D and E had higher amounts of Litsea cubeba residue added than group C, but their aerobic stability was lower than that of group C. The lower acetic acid content in groups D and E compared to group C is because the excessive amount of Litsea cubeba residue added inhibited the activity of heterofermentative bacteria that produce acetic acid.

[0037] 3. Rumen in vitro fermentation characteristics Four Jinjiang cattle with permanent rumen fistulas were selected as rumen fluid donors. Each animal weighed 365±27 kg and was housed in individual pens. The experimental diet (DM basal) consisted of 79.65% rice straw, 10.63% corn, 1.44% wheat bran, 3.63% soybean meal, 3.39% cottonseed meal, 0.09% dicalcium phosphate, 0.27% limestone powder, 0.45% salt, and 0.45% premix. The nutritional levels were CP 11.76%, NDF 60.02%, ADF 35.72%, and maintenance net energy (NEm) 3.13 MJ / kg. The animals were fed twice daily in equal amounts (08:00 and 18:00) with free access to water.

[0038] Five groups of whole-plant corn silage with different amounts of Litsea cubeba residue were blanched in a 105℃ forced-air drying oven for 20 min, and then dried in a 65℃ oven for 48 h. The dried samples were removed, and 2 g ± 0.0001 g of the dried whole-plant corn silage was weighed and added to pre-weighed nylon bags, which were then tied tightly with rubber bands. Three replicates were set up for each cow at each time point. Nylon bags with the same degradation time were tied to a nylon rope, and one end of all the ropes was tied to a mesh bag, which was then placed inside the rumen, with the ropes of the mesh bag fixed to the outside of the rumen fistula. The nylon bags containing silage were removed at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 72 h after placement, and rinsed three times in a washing machine at medium speed for 5 min each time, changing the water once in between. If a washing machine was unavailable, the bags could be rinsed by hand in cold water, gently stroking them without rubbing, until the water ran clear. The rinsed nylon bags were placed in an oven at 55°C for 48 hours until constant weight, and then weighed after 24 hours of rehydration. The residue in the nylon bags was removed, passed through a 40-mesh sieve, and stored at 4°C for later analysis of nutritional components. The contents of DM, CP, NDF, and ADF in the samples at each time point were determined. The rumen degradation parameters were calculated using the rumen dynamics mathematical model proposed by Ørskov et al. (1979), with the following formula: p = a + b (1 - e -ct In the formula: p is the degradation rate (%) of the nutrient components of the feed under test in the rumen during culture time t; a is the rapidly degraded fraction (%); b is the slowly degraded fraction (%); c is the degradation rate of the slowly degraded fraction (%) / h; t is the rumen culture time (h). Kp (outflow rate) = 0.045h -1 The effective degradation rate (ED) is calculated using the following formula: ED = a + b[c / (c + Kp)], and the results are shown in Table 3.

[0039] Table 3

[0040] Note: Different lowercase letters in the upper right corner of the data indicate significant differences between groups. P <0.05, with the same lowercase letter indicating no significant difference between groups ( P >0.05).

[0041] Table 3 shows that the rapidly degraded fraction (a) of dry matter in each treatment group ranged from 31.8% to 36.7%, the slowly degraded fraction (b) ranged from 41.2% to 46.5%, the degradation rate (c) of the slowly degraded fraction ranged from 4.17% / h to 4.60% / h, and the effective degradation rate (ED) ranged from 62.3% to 66.0%. Group E had the highest effective degradation rate of DM (66.0%), while Group C had the lowest (62.3%). There were no significant differences in the values ​​of DM a (P=0.145), b (P=0.515), c (P=0.446), and ED (P=0.262) among the groups (P>0.05), indicating that the overall availability of dry matter was relatively similar across the groups.

[0042] The a-value of the rapidly degraded CP in each treatment group remained stable between 31.9% and 33.3%, the b-value of the slowly degraded CP ranged between 30.9% and 34.2%, and the degradation rate c-value of the slowly degraded CP ranged between 4.63% / h and 4.83% / h. The calculated effective degradation rate (ED) of CP ranged from 54.2% to 55.4%, corresponding to a rumen-protected protein (RUP) content of 44.6% to 45.8%. There were no significant differences in the a-value (P=0.843), b-value (P=0.420), c-value (P=0.954), and ED-value (P=0.610) of CP among the treatment groups (P>0.05). This indicates that the crude protein availability was relatively similar across the treatment groups.

[0043] For neutral detergent fiber (NDF), the rapid degradation fraction (a) in the control group was 4.28%, with no significant difference in the rapid degradation fraction among the treatment groups (P>0.05). The slow degradation fraction (b) in the control group was 75.75%, with no significant difference in the slow degradation fraction among the treatment groups (P>0.05). The slow degradation rate (c) in the control group was 3.16%, with no significant difference in the slow degradation rate among the treatment groups (P>0.05). The effective degradation rate (ED) in the control group was 50.2%, with no significant difference in the effective degradation rate among the treatment groups (P>0.05). For acid detergent fiber (ADF), the rapid degradation fraction (a) in the control group was 2.21%, with no significant difference in the rapid degradation fraction among the treatment groups (P>0.05). The slow degradation fraction (b) in the control group was 74.45%, with no significant difference in the slow degradation fraction among the treatment groups (P>0.05). The slow degradation rate (c) of the control group was 2.70% / h, and there was no significant difference in the slow degradation rate of ADF among the treatment groups (P>0.05). The effective degradation rate (ED) of the control group was 47%, and there was no significant difference in the effective degradation rate of ADF among the treatment groups (P>0.05).

[0044] 4. Aerobic stability When the above five groups of mixed raw materials with different amounts of Litsea cubeba residue were ensiled and fermented for 60 days, 150 g of silage was placed in a clean 500 mL wide-mouth bottle and placed in an insulated and heat-resistant place. A sensitive mercury thermometer was inserted into the center of the silage to measure the temperature change, while the room temperature was monitored. The time (h) from the time the sample came into contact with air until the sample temperature was 2°C higher than the room temperature was defined as the duration of aerobic stability. The average air temperature during the aerobic exposure period was 22.6°C, and the average humidity was 81%. The results are shown in Table 4.

[0045] Table 4

[0046] Note: Different lowercase letters in the upper right corner of the data indicate significant differences between groups (P<0.05), while the same lowercase letter indicates no significant differences between groups (P>0.05).

[0047] As shown in Table 4, Group C (with 2% Litsea cubeba residue) had the best aerobic stability effect, and the duration of aerobic stability was significantly longer than that of other groups (P<0.05).

[0048] In summary, as shown in Tables 1-4, adding 2% of the fresh weight of the whole corn plant with Litsea cubeba residue results in better silage performance. Therefore, 2% is selected as the optimal addition amount of Litsea cubeba residue.

[0049] 5. Dynamic changes in silage metabolism and microorganisms during aerobic exposure. The mixed raw material with 2% Litsea cubeba residue added in Example 1 was ensiled for 60 days and then subjected to aerobic exposure. The aerobic exposure conditions were: ambient temperature 22.6℃, ambient humidity 81%, and the samples were placed in open trays and stored in the dark. Samples were taken at 0 days (when opened), 2 days, and 5 days of aerobic exposure, and approximately 200 g of silage samples were immediately flash-frozen in liquid nitrogen and then transferred to -80℃ for storage. The samples were transported to the biotechnology company using dry ice for metabolomics and 16S rDNA sequencing analysis. The sequencing experimental procedure is as follows: Figure 1As shown. The bioinformatics analysis workflow is as follows: To ensure the accuracy of OTU (Operational Taxonomic Unit) clustering and subsequent analysis, the raw sequencing data is first filtered to obtain valid data. Then, based on the valid data, OTU clustering / denoising and species classification analysis are performed to form species abundance profiles at OTU and other species classification levels. Based on the OTU species abundance profiles after data homogenization, OTU abundance, diversity index, and other analyses are performed, and statistical analysis of community structure is conducted on species annotations at each classification level. Based on the above analysis, a series of cluster analyses and statistical comparative analyses based on OTU and species composition are performed to explore differences in species composition between samples. Correlation statistical analysis is conducted in conjunction with environmental and clinical factors to identify significantly related species communities and perform functional prediction analysis of microbial communities, such as... Figure 2 As shown.

[0050] It is important to note that the bioinformatics analysis workflow uses the DADA2 method recommended by QIIME2 for noise and chimerism removal. Compared to the results obtained by clustering methods such as UPARSE in QIIME1, DADA2 provides more accurate results than the previous generation. Because clustering is no longer based on similarity, the generated representative sequence is no longer an OTU. More precisely, in this invention, OTU should be referred to as an amplicon sequence variant (ASV) or feature sequence. However, for ease of understanding, this invention uses OTU to represent the representative sequence of this amplicon feature, which is also the common practice among researchers in the field of amplicon research.

[0051] Association analysis combining metabolomics and 16S rDNA sequencing revealed the underlying mechanisms related to the aerobic stability of whole-plant maize silage. Specific findings are as follows: (1) Enrichment of key antibacterial and antioxidant substances Non-targeted metabolomics analysis by LC-MS (as attached) Figure 3 As shown in the figure, the content of specific metabolites in silage was significantly higher in the treatment group than in the control group. Among them, (+)-Magnoflorine, a natural apophene-type isoquinoline alkaloid, showed extremely high relative abundance in the treatment group. This substance has significant anti-inflammatory (inhibition of pro-inflammatory factors such as TNF-α and IL-6) and antioxidant (scavenging free radicals) properties. In addition, random forest trait importance analysis (as shown in the attached figure) Figure 4(As shown) further confirms that Malabaricone is the key characteristic variable distinguishing the treatment group from the control group. Malabaricone is a class of diarylnonane natural organic compounds with strong antibacterial and antifungal activities, effectively inhibiting the growth of Aspergillus flavus and scavenging DPPH and ABTS. + Free radicals. The significant increase in the content of these two key active substances in the treatment group constitutes the material basis for improving the aerobic stability of whole-plant silage maize.

[0052] (2) Response of differential metabolic pathways KEGG pathway ORA enrichment analysis (see attached) Figure 5 As shown in the figure, compared with the control group, the phenylpropanoid biosynthesis pathway had the highest complexation ratio (0.077) and the most significant p-value (red), indicating that this pathway is the core pathway with the highest enrichment of differential metabolites. 2-Oxocarboxylic acid metabolism, amino acid biosynthesis, and phenylalanine metabolism also showed high complexation ratios (>0.06) and significant p-values ​​(red-orange). It is evident that differential metabolites are mainly enriched in phenylpropanoid and amino acid biosynthesis and degradation pathways (such as phenylalanine and tryptophan metabolism). Microbial metabolism may be affected by stress or environmental regulation, involving biological processes such as energy synthesis, antioxidant defense, or secondary metabolite accumulation. The activation of these pathways indicates that the addition of Litsea cubeba regulates the accumulation of secondary metabolites during silage, enhancing the system's antioxidant defense and energy metabolism capabilities.

[0053] (3) Dynamic succession of microbial communities Regarding bacterial communities (as shown in the appendix) Figure 6 and 7As shown in the figure, at the phylum level, Firmicutes (Bacillota) and Pseudomonadota are the dominant bacterial phyla, playing important roles in hydrolysis and acid production. With increasing silage exposure time, the abundance of Firmicutes decreases, while the abundance of Pseudomonadota increases. At the genus level, *Acetobacter*, *Lactobacillus*, *Paenibacillus*, and *Limosilactobacillus* are the dominant bacterial genera. With increasing aerobic exposure time, the abundance of *Lactobacillus* decreases, while the abundance of *Acetobacter* increases significantly. *Acetobacter* can oxidize lactic acid and acetic acid into carbon dioxide and water, and is an important bacterium leading to silage spoilage. Compared with the control group, the experimental group showed a significantly higher level of Bacillus species. Bacillus species can significantly inhibit the growth of aerobic microorganisms (such as molds) after silage is opened and exposed to air through its strong aerobic metabolic capacity and the ability to produce antibacterial substances, thereby greatly improving the aerobic stability of silage corn.

[0054] Regarding fungal communities (as shown in the appendix) Figure 8 and 9 As shown in the figure, at the phylum level, Ascomycota and Basidiomycota are the dominant fungal phyla, with Ascomycota having a particularly high proportion, covering more than 90% of the observed total sequences. At the genus level, *Aspergillus* and *Cladosporium* are the dominant fungal genera in all groups. *Aspergillus* and *Cladosporium* are very important and extremely widespread fungal genera in Ascomycota, and are pathogens that cause mold in crops. Figure 9 It can be seen that the relative abundance of Aspergillus and Cladosporium in the experimental group was lower than that in the control group because the addition of Litsea cubeba residue inhibited the growth of these harmful bacteria and improved the aerobic stability of silage corn.

[0055] In summary, by adding Litsea cubeba residue to introduce active ingredients such as (+)-magnoliaine and malabathone, the bacteria directly exert antibacterial and antioxidant effects while regulating metabolic pathways such as phenylpropanoids. At the microecological level, it promotes the growth of beneficial bacteria (such as Bacillus subtilis) and significantly inhibits the proliferation of putrefactive bacteria (such as Acetobacter) and mold-causing bacteria (such as Aspergillus and Cladosporium). This dual regulation mechanism of substances and microorganisms clarifies the key factors affecting the aerobic stability of whole-plant maize, providing a solid theoretical basis for targeted improvement of silage quality and ensuring the safety of livestock feed, thus promoting the rapid development of animal husbandry.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the quality of whole-plant maize silage using Litsea cubeba residue, characterized in that, Includes the following steps: (1) After crushing the whole corn plant, add the Litsea cubeba residue to obtain a mixed raw material, wherein the amount of Litsea cubeba residue added is 1%-4% of the fresh weight of the crushed whole corn plant; (2) The mixed raw materials are loaded into a silage container, compacted and sealed, and fermented to obtain the whole-plant corn silage.

2. The method for improving the quality of whole-plant maize silage using Litsea cubeba residue according to claim 1, characterized in that, The Litsea cubeba residue mentioned in step (1) is the residue remaining after extracting essential oil from Litsea cubeba fruit.

3. The method for improving the quality of whole-plant maize silage using Litsea cubeba residue according to claim 1, characterized in that, The amount of Litsea cubeba residue added in step (1) is 2% of the fresh weight of the whole corn plant after crushing.

4. The method for improving the quality of whole-plant maize silage using Litsea cubeba residue according to claim 1, characterized in that, The silage fermentation described in step (2) is carried out for 60 days.

5. The method for improving the quality of whole-plant maize silage using Litsea cubeba residue according to claim 1, characterized in that, After the silage fermentation described in step (2) is completed, aerobic exposure is carried out for 0-5 days.

6. The method for improving the quality of whole-plant maize silage using Litsea cubeba residue according to claim 1, characterized in that, The quality of whole-plant corn silage includes aerobic stability, nutritional components, fermentation quality, and rumen in vitro fermentation characteristics.

7. The method for improving the quality of whole-plant maize silage using Litsea cubeba residue according to claim 6, characterized in that, The method for determining the nutritional components and rumen in vitro fermentation characteristics is as follows: the whole-plant corn silage is blanched and then dried, and then the nutritional components and rumen in vitro fermentation characteristics are determined.

8. The method for improving the quality of whole-plant maize silage using Litsea cubeba residue according to claim 6, characterized in that, The method for determining the fermentation quality is as follows: the silage after aerobic exposure is made into an extract, and the pH and fermentation quality are measured.

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